The present invention relates to liver-related transcription factors and, in particular, to such factors as participate in the regulation of a variety of genes such as certain of the apolipoproteins involved in fat and cholesterol transport.
This invention also relates to antibodies which recognize the receptor HNF-4, and antiidiotype antibodies that recognize both antibodies-to HNF-4 and ligands which bind to HNF-4.
The invention also relates to antisense DNA and RNA molecules complementary to mRNA for HNF-4, and ribozymes which recognize the mRNA.
The invention also relates to methods of use of the aforementioned molecules, DNA sequences, antibodies, anti-idiotype antibodies, antisense molecules and ribozymes, for example in developing diagnostic and therapeutic agents to detect, inhibit or enhance binding to HNF-4.
It is a principal object of this invention to provide new means to study, diagnose, prevent and treat disease. More particularly, it is an object of this invention to provide molecules involved in binding to HNF-4, and to isolate other molecules which are themselves useful in inhibiting such binding.
This invention provides DNA sequences that code on expression for HNF-4, genomic DNA sequences for HNF-4, recombinant DNA molecules containing these DNA sequences, unicellular hosts transformed with these DNA molecules, processes for producing such receptors, and proteins essentially free of normally associated animal proteins.
The present invention also provides for antibody preparations reactive for HNF-4.
Monoclonal antibodies recognizing ligands to HNF-4 can inhibit ligand binding directly or by binding or otherwise interacting with a third molecule. Such molecules may act, for example, by changing the surface conformation of the ligand so that its affinity for the HNF-4 is reduced.
This invention also provides recombinant DNA molecules containing HNF-4DNA sequences and unicellular hosts transformed with them. It also provides for HNF-4 proteins essentially free of normally associated animal proteins, methods for producing HNF-4, and monoclonal antibodies that recognize HNF-4.
This invention further provides methods for using antisense nucleic acids and ribozymes to inhibit HNF-4 expression. The invention also relates to methods for identifying binding inhibitors by screening molecules for their ability to inhibit binding of HNF-4 to its ligand. It provides methods for identifying ligands. One such method involves using anti-idiotypic antibodies against antibodies that recognize HNF-4 or HNF-4 ligands.
Cell type specificity is based on differential gene expression which is in turn determined, at least in part, by the particular set of transcription factors present and active in a given cell at a given time. Many such factors have been identified and characterized, particularly in the liver where there is a wide range of transcriptionally controlled genes (McKnight and Palmiter, 1979; Derman et al., 1981). Some transcription factors, such as AP-1 and Sp-1, seem to be present in all cells at all times but other factors have a more limited distribution. Whether there is a discernible logic that explains the distribution of the many factors has yet to be determined. Two aspects of this problem are particularly important. The first aspect is to determine whether the distribution of factors in different issues is controlled at the level of transcription. If so, then a cascade of transcriptional regulation that ultimately results in cell specificity is indicated. The second issue is whether any particular factor is central to the accomplishment of a particular metabolic or physiologic goal. Such a goal might be suggested by factors acting on an interrelated set of genes.
These issues have begun to be addressed by the dissection and analysis of the promoter/enhancer regions of genes expressed primarily in hepatocytes by the present applicants and others (Johnson, 1990). The DNA elements that confer cell specific expression have been defined by transient transfection into cultured cells (e.g., hepatoma vs. HeLa cells) and/or in vitro transcription assays, and the proteins that bind to these elements have been identified by DNA binding assays using crude liver nuclear extracts. In this way, at least four distinct protein factors that are abundant in liver have been found thus far: HNF1 (LF-B1) (Courtois et al., 1987; Monaci et al., 1988), C/EBP (Johnson et al., 1987), HNF-3 and HNF-4 (Costa et al., 1989). HNF1, a homeo-domain protein (Frain et al., 1989; Baumhueter et al., 1990), C/EBP, the original leucine zipper protein (Landschulz et al., 1988), and most recently HNF-3A, a DNA binding protein that has no similarity to known transcription factor families (Lai et al., 1990) have all been purified and cloned so that distribution and regulation of each can be determined.
The following publications are cited in the body of the patent application. Each of the publications is incorporated herein by reference:
Ahe, von der D., Janich, S., Scheidereit, C., Renkawitz, R., Schutz, G., and Beato, M. (1985). Glucocorticoid and progesterone receptors bind to the same sites in two hormonally regulated promoters. Nature, 313, 706-709.
Baumhueter, S., Mendel, D. B ., Conley, P. B., Kuo, C. J., Turk, C., Graves, M. K., Edwards, C. A., Courtois, G., and Crabtree, G. R. (1990). HNF-1 shares three sequence motifs with the POU domain proteins and is identical to LF-B1 and APF. Genes and Development 4, 372-379.
Beato, M. (1989). Gene regulation by steroid hormones. Cell 56, 335-344.
Birkenmeier, E. H., Gwynn, B., Howard, S., Jerry, J., Gordon, J. I., Landsdhulz, W. H., and McKnight, S. L. (1989). Tissue-specific expression, developmental regulation and mapping of the gene encoding CCAAT/enhancer binding protein. Genes and Development, 3, 1146-1156.
Brand, N., Petkovich, M., Krust, A., Chambon, P., de The, H., Marchio, A., Tiollais, P., and Dejean, A. (1988). Identification of a second human retinoic acid receptor. Nature, 332, 850-853.
Breslow, J. (1988). Apolipoprotein genetic variation and human disease. Physiol. Reviews, 68, 85-132.
Capon, D. J. et al. (1989). Designing CF4 immunoadhesins for AIDS therapy. Nature, 337, 525-531.
Carlsson, R., and Glad, C. (June, 1989). Monoclonal antibodies into the ""90s. Bio/Techblogy, 7, 567-573.
Cate, R. et al. (1986). Isolation of the bovine and human genes for Mullerian inhibiting substance and expression of the human gene in animal cells. Cell, 45, 685-598.
Cech, T. R. (1988). Ribozymes and their medical implications. J. Amer. Med. Assn., 260, 3030-3044.
Chomezynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156-159.
Costa, R. H., Lai, E., and Darnell, J. E., Jr. (1986). Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific. Mol. and Cell. Biol., 6, 4697-4708.
Costa, R. H., Grayson, D. R., Xanthopoulos, K. G., and Darnell, J. E., Jr. (1988). A liver-specific DNA-binding protein recognizes multiple nucleotide sites in regulatory regions of transthyretin, a1-antitrypsin, albumin, and simian virus 40 genes. Proc. Natl. Acad. Sci., 85, 3840-3844.
Costa, R. H., Grayson, D. R. and Darnell, J. E. Jr. (1989). Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and al-antitrypsin genes. Mol. and Cell. Biol., 9, 1415-1425.
Costa, R. H., Van Dyke, T. A., Yan, C., Kuo, F., and Darnell, J. E., Jr. (1990). Similarities in transthyretin gene expression and differences in transcription factors: liver and yolk sac compared to choroid plexus. Proc. Natl. Acad. Sci. USA, 87, 6589-6593.
Courtois, G., Morgau, J. G., Campbell, L. A., Fourel, G. and Crabtree, G. R. (1987). Interaction of a liver-specific nuclear factor with the fibrinogen and al-antitrypsin promoters. Science, 238, 688-692.
Danielsen, M., Hinck, L., and Ringold, G. M. (1989). Two amino acids within the knuckle of the first zinc finger specify DNA response element activation by the glucocorticoid receptor. Cell, 57, 1131-1138.
Davis, M. M. (1986). Subtractive cDNA hybridization and the T-cell receptor gene. Handbook of Experimental Immunology in Four Volumes, 4th ed. Blackwell Scientific Publications, Oxford, England, 76.1-76.13.
Davis, M. M. et al. (1984). Cell type-specific cDNA probes and the murine I region: The localization and orientation of ad. Proc. Natl. Acad. Sci. USA, 81, 2194-2198.
Derman, E., Krauter, K., Walling, L., Weinberger, C., Ray, M. and Darnell, J. E. Jr. (1981). Transcriptional control in the production of liver-specific mRNAs. Cell, 23, 731-739.
de The, H., Marchio, A., Tiollais, P., and Dejean, A. (1987). A novel steroid thyroid hormone receptor-related gene inappropriately expressed in human hepatocellular carcinoma. Nature, 330, 667-670.
Duguid, J. R. et al. (1988). Isolation of cDNAs of scrapie-modulated RNMAs by subtractive hybridization of a cDNA library. Proc. Natl. Acad. Sci. USA, 85, 5738-5742.
Evans, R. M. (1988). The steroid and thyroid hormone receptor superfamily. Science, 240, 889-895.
Fawell, S. E., Lees, J. A., White, R. and Parker, M. G. (1990). Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor. Cell, 60, 953-962.
Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132, 6-13.
Fisher, R. A. et al. (1988). HIV infection is blocked in vitro by recombinant soluble CD4. Nature, 331, 76-78.
Forman, B. M., Yan, C. R., Au, M., Casanova, J., Ghysdael, J., and Samuels, H. H. (1989). A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors. Mol. End., 3, 1610-1626.
Forman, B. M. and Samuels, H. H. (1990). Interactions among a subfamily of nuclear hormone receptors: The regulatory zipper model. Mol. End. 4, 1293-1301.
Frain, M., Swart, G., Monaci, P., Nicosia, A., Stampfli, S., Frank, R., and Cortese, R. (1989). The liver-specific transcription factor LF-Bl contains a highly diverged homeobox DNA binding domain. Cell, 59, 145-157.
Fried, M. and Crothers, D. M. (1981). Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res., 9, 6505-6525.
Giguere, V., Yang, N., Segui, P., and Evans, R. M. (1988). Identification of a new class of steroid hormone receptors. Nature, 331, 91-94.
Glass, C. K., Lipkin, S. M., Devary, O. V., and Rosenfeld, M. G. (1989). Positive and negative regulation of gene transcription by a retinoic acid-thyroid hormone receptor heterodimer. Cell, 59, 697-708.
Gorman, C. M., Moffat, L. F. and Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol., 2, 1044-1051.
Gorman, C. M., Howard, B. H. and Reeves, R. (1983). Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res., 11, 7631-7648.
Gorski, K., Carneiro, M. and Schibler, U. (1986). Tissue-specific in vitro transcription from the mouse albumin promoter. Cell, 47, 767-776.
Green, N., Alexander, H., Olson, A., Alexander, S., Shinnick, T. M., Sutcliffe, J. G., and Lerner, R. A. (1982). Immunogenic structure of the influenza virus hemagglutinin. Cell, 28, 477-487.
Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J. M., Argos, P., and Chambon, P. (1986). Human oestrogen receptor cDNA: Sequence, expression and homology to c/epb. Nature, 320, 134-139.
Green, S. and Chambon, P. (1988). Nuclear receptors enhance our understanding of transcription regulation. Trends Genet., 4, 309-314.
Gubler, U., and Hoffman, B. J. (1983). A simple and very efficient method for generating cDNA libraries. Gene, 25, 263-269.
Hamada, K., Gleason, S. L., Levi, B. Z., Hirschfeld, S., Appella, E., and Ozato, K. (1989). H-2 RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element. Proc. Natl. Acad. Sci. USA, 86, 8289-8293.
Hambor, J. E. et al. (1988). Functional consequences of antisense RNA-mediate inhibition of CD8 surface expression in a human T cell clone. J. Exp. Med., 168, 1237-1245.
Hardon, E. M., Frain, M., Paonessa, G. and Cortese, R. (1988). Two distinct factors interact with the promoter regions of several liver-specific genes. The EMBO J., 7, 1711-1719.
Harlow, E. and Lane, D. (1988). Antibodies: A laboratory manual. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Lab.)
Hasselhoff, J., and Gerlach, W. L. (1988). Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature, 334, 585-591.
Hedrick, S. M. et al. (1984). Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature, 308, 149-153.
Ito, Y., Azrolan, N., O""Connell, A., Walsh, A., and Breslow, J. L . (1990) Hypertriglyceridemia as a result of human apoCIII gene expression in transgenic mice. Science, 249, 790-793.
Johnson, P. F., Landschulz, W. H., Graves, B. J., and McKnight, S. L. (1987). Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses. Genes and Development, 1, 133-146.
Johnson, P. F. (1990). Transcriptional activators in hepatocytes. In Cell Growth and Differentiation, 1, 47-52.
Kadonaga, J. T., and Tjian, R. (1986). Affinity purification of sequence-specific DNA binding proteins. Proc. Natl. Acad. Sci. USA, 83, 5889-5893.
Kennedy, R. C. et al. (July, 1986). Anti-idiotypes and immunity. Sci. Am., 255, 48-56.
Kozak, M. (1987). An analysis of 5xe2x80x2-noncoding sequences from 699 vertebrate messenger RNA""s. Nucleic Acids Res., 15, 8125-8143.
Krebs, E., Eisenman, R., Kuenzel, E., Litchfield, D., Lozeman, F., Lischer, B. and Sommercorn, J. (1988). Casein kinase II as a potentially important enzyme concerned with signal transduction. In Molecular Biology of Signal Transduction. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), p. 77-84.
Kumar, V., and Chambon, P. (1988). The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell, 55: 145-156.
Kuo, C. F., Xanthopoulos, K. G., and Darnell, J. E. Jr. (1990). Fetal and adult localization of C/EBP: evidence for combinatorial action of transcription factors in cell-specific gene expression. Development, 109, 473-481.
Lai, E., Prezioso, V. R., Smith, E., Litvin, O., Costa, R. H., and Darnell, J. E., Jr. (1990). HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally. Genes and Development, 4, 1427-1436.
Landschulz, W. H., Johnson, P. F., Adashi, E. Y., Graves, B. J., and McKnight, S. L. (1988). Isolation of a recombinant copy of the gene encoding C/EBP. Genes and Development, 2, 786-800.
Lathe, E. (1985). Synthetic oligonucleotide probes deduced from amino acid sequence data: Theoretical and practical considerations. J. Mol. Biol., 183, 1-12.
Leff, T., Reue, K., Melian, A., Culver, H., and Breslow, J. L. (1989). A regulatory element in the ApoCIII promoter that directs hepatic specific transcription binds to proteins in expressing and nonexpressing cell types. The J. of Biol. Chem., 264, 16132-16137.
Lew, D. J., Decker, T., Strehlow, I. and Darnell, J. E. (1990). Overlapping elements in the GBP gene promoter mediate transcriptional induction by alpha and gamma-interferon. Mol. Cell Biol., in press.
Li, Y., Shen, R.-F., Tsai, S. Y., and Woo, S. L. C. (1988). Multiple hepatic trans-acting factors are required for in vitro transcription of the human alpha-1-antitrypsin gene. Mol. and Cell. Biol., 8, 4362-4369.
MacGregor, G. R., and Caskey, C. T. (1989). Construction of plasmids that express E. coli b-galactosidase in mammalian cells. Nucleic Acids Res., 17, 2365.
Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989). Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive element. Nature, 338, 271-274.
Mangelsdorf, D. J., Ong, E. S., Dyck, J. A. and Evans, R. M. (1990). Nuclear receptor that identifies a novel retinoic acid response pathway. Nature, 345, 224-229.
Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular cloning: A laboratory manual. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Lab.)
Marcus-Sekura, C. J. (1988). Techniques for using antisense oligonucleotides to study gene expression. Anal. Biochem., 172, 289-295.
Matsudaira, P. (1987). Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluorid membranes. The J. of Biol. Chem., 262, 10035-10038.
McKnight, G. S., and Palmiter, R. D. (1979). Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J. Biol. Chem., 254, 9050-9058.
Mermod, N., O""Neill, E. A., Kelly, T. J. and Tjian, R. (1989). The proline-rich transcriptional activator of CTF/NF-1 is distinct from the replication and DNA binding domain. Cell, 58, 741-753.
Miyajiima, N., Kadowaki, Y., Fukushige, Shiminizu, S., Semba, K., Yamanashi, Y. H., Matsubara, K., Toyoshima, K., and Yamanoto, T. (1988). Identification of two novel members of erbA superfamily by molecular cloning: The gene products of the two are highly related to each other. Nucleic Acids Res., 16:11057-11074.
Monaci, P., Nicosia, A., and Cortese, R. (1988). Two different liver-specific factors stimulate in vitro transcription from the human a1-antitrypsin promoter. The EMBO J., 7, 2075-2087.
Mueller, C. R., Maire, P., and Schibler, U. (1990). DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally. Cell, 61, 279-291.
Pearson, W. R., and Lipman, D. J. (1988). Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA, 85, 2444-2448.
Puissant, C., and Houdebine, L. M. (1990). An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. BioTachniaues, 8, 148-149.
Reue, K., Leff, T., and Breslow, J. L. (1988). Human apolipoprotein CIII gene expression is regulated by positive and negative Cis-acting elements and tissue-specific protein factors. The J. of Biol. Chem., 263, 6857-6864.
Rosen, C. A., Sodroski, J. G. and Haseltine, W. A. (1985). The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell, 41, 813-823.
Ruppert, S., Boshart, M., Bosch, F. X., Schmid, W., Fournier, R. E. K., and Schutz, G. (1990). Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes. Cell, 61, 895-904.
Ryseck, R. P., Macdonald-Bravo, H., Mattei, M. G., Ruppert, S., and Bravo, R. (1989). Structure, mapping and expression of a growth factor inducible gene encoding a putative nuclear hormonal binding receptor. The EMBO J., 8, 3327-3335.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491.
Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74, 5463-5467.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning: A laboratory manual. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory).
Sargent, T. D. (1987). Isolation of differentially expressed genes. Methods in Enzymol., 152, 423-447.
Schule, R., Umesono, K., Mangelsdorf, D. J., Bolado, J., Pike, J. W., and Evans, R. M. (1990). Jun-fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene. Cell, 61, 497-504.
Seed, B. (1987). An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature, 329, 840-842.
Seed, B., and Aruffo, A. (1987). Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. USA, 84, 3365-3369.
Tsai, S. Y., Carlstedt-Duke, J., Weigel, N. L., Dahlman, K., Gustafsson, J. A., Tsai, M. J., and O""Malley, B. W. (1988). Molecular interactions of steroid hormone receptor with its enhancer element: Evidence for receptor dimer formation. Cell, 55, 361-369.
Umesono, K., Giguere, V., Glass, C. K., Rosenfeld, M. G., and Evans, R. M. (1988). Retinoic acid and thyroid hormone induce gene expression through a common responsive element. Nature, 336, 262-265.
Umesono, K. and Evans, R. M. (1989). Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell, 57, 1139-1146.
Vaulont, S., Puzenat, N., Kahn, A., and Raymondjean, M. (1989). Analysis by cell-free transcription of the liver-specific pyruvate kinase gene promoter. Mol. and Cell. Biol., 9, 4409-4415.
Wang, L. H., Tsai, S. Y., Cook, R. G., Beattie, W. G., Tsai, M. J. and O""Malley, B. W. (1989). COUP transcription factor is a member of the steroid receptor superfamily. Nature, 340, 163-166.
Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruo, D. J., and Evans, R. M. (1986). The c/epb gene encodes a thyroid hormone receptor. Nature, 234, 641-646.
Wingender, E. (1990). Transcription regulating proteins and their recognition sequences. Critical Reviews in Eukarvotic Gene Expression, 1, 11-48.
Wysocki, L. J., and Sato, V. L. (1978). Panning for lymphocytes: A method for cell selection. Proc. Natl. Acad. Sci. USA, 75, 2844-2848.
Xanthopoulos, K. G., Mirkovitch, J., Decker, T., Kuo, C. F., and Darnell, J. E., Jr. (1989). Cell-specific transcriptional control of the mouse DNA-binding protein mC/EBP. Proc. Natl. Acad. Sci. USA 86, 4117-4121.
Yamasaki, K. et al. (1988). cloning and expression of the human interleukin-6 (BSF-2/IFNB2) receptor. Science, 241, 825-828.
Young, R. A. and Davis, R. W. (1983). Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA, 80, 1194-1198.
The present invention comprises the purification and cloning of HNF-4 (hepatocyte nuclear factor 4), a factor originally detected in crude liver extracts as binding to a DNA element required for the transcription of the transthyretin (TTR) gene in hepatoma cells (Costa et al., 1989). An amino acid sequence comparison indicates that HNF-4 is a member of the superfamily of steroid/thyroid hormone receptors, ligand-dependent transcription factors which are known to play a role in differentiation and development (Evans, 1988; Green and Chambon, 1988; Beato, 1989). Whereas all of the other members to date fall into one of several subfamilies based on the nucleotide sequence of their recognition sites and the amino acid sequence of the zinc finger region (Umesono and Evans, 1989; Forman and Samuels, 1990), HNF-4 appears to represent a new subfamily.
More particularly, the present transcription factor is believed to play a regulatory role in the formation of lipid carrying proteins such as Apo CIII, as well as possible effects on Apo A1, Apo B, pyruvate kinase, xcex11 antitrypsin and glutamine synthetase. The cDNA sequence has been identified, and the invention relates to the DNA sequence, recombinant molecules based thereon, probes, sense and antisense RNA, and appropriately transformed host cells. Diagnostic and therapeutic applications are likewise contemplated.